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Primer Design Tips for Seamless PCR Cloning

Interested in trying seamless PCR cloning but don’t know where to start? Or maybe you just need a quick refresher? Here’s a list of top tips to keep in mind when designing your primers for seamless cloning, including some information specific to In-Fusion Cloning.

  1. The 3′ end of the primer should contain sequence that is specific to the target gene you are amplifying. This sequence should be 18–25 bases long and should ideally have a GC content between 40–60%.

  2. Most seamless cloning protocols, including the Gibson method, require the 5′ end of the primer to contain 25 bases that are homologous to one end of the DNA fragment to which it will be joined (i.e., the vector, or another insert). If you are using In-Fusion Cloning, this overlap can be shortened to 15 bases for single-insert cloning and 20 bases for multiple-insert cloning.

  3. Aim for melting temperatures (Tms) between 58–65°C. (See Tip 4 for details on calculating the Tm.) If the difference between the Tms for your forward and reverse primers is >4°C, you are not as likely to get good amplification.

  4. The primer Tm is calculated from the 3′ (gene-specific) end of the primer, NOT the entire primer. If the Tm is too low, increase the length of the gene-specific portion to reach a Tm between 58–65°C.

  5. NOTE: During PCR amplification, the first-cycle Tm should be based on just this 3′ portion of the primer. All remaining cycles should be based on the full-primer Tm.

  6. Things to avoid:
    1. Runs of identical nucleotides in the 3′ portion of the primer. The last five nucleotides at the 3′ end should contain no more than two guanines or cytosines.
    2. Complementarity within your primers to prevent hairpin structures.
    3. Complementarity between primer pairs to avoid primer dimers.

  7. Check for primer specificity.
    1. You can perform a BLAST search to see if the 3′ portion of your primer might hybridize to unintended sites on your template.
    2. LALIGN is another free sequence alignment tool that can help you identify potential unintended targets within your template sequence.

  8. Primer quality depends on the vendor and can vary from lot to lot. If the quality of your primer is poor (e.g., has many premature termination products), or is longer than 45 nucleotides, a PAGE-purification step may be necessary. In most cases, desalted oligonucleotide primers work just fine.

  9. Regardless of your specific primer designs, be sure to give your seamless cloning the highest probability of success by remembering these standard best practices for PCR:
    1. Before you start, ensure that you have the right materials. Always use PCR-grade water (both as a reaction component and to reconstitute your primers), fresh DNA polymerase and dNTPs, and high-quality DNA template.
    2. Do NOT use low-fidelity polymerases, such as Taq, which can introduce errors into your sequence. DO use a high-fidelity DNA polymerase to generate your insert to ensure sequence accuracy. We recommend CloneAmp HiFi PCR Premix, or PrimeSTAR Max DNA Polymerase.


    Still feel unsure? Online tools are available that can simplify primer design and reassure you that you are designing primers properly. We highly recommend our In-Fusion primer design tool for step-by-step guidance with In-Fusion Cloning, as it can help you design primers for single- or multiple-fragment cloning, or even site-directed mutagenesis.

    Looking for more information about designing projects that use In-Fusion technology? Check out our In-Fusion primer design FAQs for details on specific cloning applications.

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